Thin layer chromatographic method

ABSTRACT

A method for the chromatographic separation of substances, wherein the improvement comprises the steps dipping or wetting one edge of the surface of a non-porous solid constituting the stationary phase into or by a liquid constituting the mobile phase, and making said liquid rise by spreading on said surface as a continuous liquid film.

United States Patent Cremer et al.

THIN LAYER CHROMATOGRAPHIC METHOD Inventors: Erika Cremer. Innsbruck,Austria; Thaddaus Kraus, Vaduz, Liechtenstein Assignee: BalzersPatent-und Beteilig ungs-Aktiengesellschaft Filed: Sept. 2, 1969 Appl.No.: 854,456

[56] References Cited UNITED STATES PATENTS 3,327,857 6/1967 Kopp..210/198 C 3,477,950 11/1969 Clement et al. 10/198 C 3,530,707 9/1970Zimmermann ..210/31 X Primary Eraminer-Charles N. Hart Attorney-KeithMisegades and George R. Douglas, Jr.

[5 7] ABSTRACT A method for the chromatographic separation ofsubstances,

US. Cl ..210/31 wherein the improvement comprises the steps dipping or1m.c| ..B0ld 17/06 Wetting one edge 0f the surface of a non-porous SolidField of Search ..210/31 c, 198 c inning the animal? Phase by a quidconstituting the mobile phase, and making said liquid rise by spreadingon said surface as a continuous liquid film.

13 Claims, 4 Drawing figures 4 l l e 6/ 5 P A n n 3 2 i; 1

PATENTEnJuu 13 m2 SHEET 10F 2 FIG-Z ERIKA CREMER and THADDAUS KRAUSINVENTOR 5 BY MISEGADES 8c DOUGLAS ATTORNEY P'A'TENTEDJun 12 m2 SHEET 2BF 2 all/4% 35 FIG. 4.

THIN LAYER CHROMATOGRAPHIC METHOD BACKGROUND OF THE INVENTION layer of athickness of the order of magnitude of 1 mm. Occasionally layerthicknesses smaller than 1 mm have been used, but according to the viewheld generally (conf. e.g. E. STAHL Thin-Layer Chromatography, 2ndEdition, Springer Verlag 1968) a limit is reached at 0.1 mm below whichit does not make sense to go since then a marked deterioration in thedissolving capacity occurs.

Accordingly in this known thin-layer chromatography a thin layer ofporous structure is needed, the capillaries of which play a similar partas the interstices between the individual grains of a chromatographiccolumn.

BRIEF SUMMARY OF THE INVENTION The present invention has the object ofproviding a new kind of chromatography which offers advantages asregards the minimum amounts required for obtaining a chromatography ofthe substances to be analyzed, and in many cases also as regards theseparation effect by which substances of similar adsorption capacity canbe distinguished by chromatography, as well as in regard of simplicityof the apparatus required for carrying out the method and of thereduction of the time required for obtaining a chromatograph to onetenth or even to one hundredth.

The method according to the present invention for the chromatographicseparation of substances by letting a liquid flow as the mobile phaseover a solid serving as the stationary phase is characterized in that bywetting the lower edge of the surface of a non-porous solid body theliquid is made to rise by spreading as a closed liquid film.

In particular, freshly prepared rough surfaces and the surfaces of thinlayers produced by vapor deposition of a metal and subsequent oxidationat elevated temperatures in air have proved effective. Such surfacesshow a so-called specific (true) surface exceeding considerably themacroscopic (apparent) surface.

An advantage of the use of vapor deposited layers in thinlayerchromatography consists in the complete homogeneity of the layers. Bystandardizing the vapor deposition process layers of identicalproperties can be produced, and thus accurately reproducible separationresults can be attained. Such layers have great mechanical stability andcan be used repeatedly since they can be regenerated after use simply byrinsing, boiling or annealing in air at 400700 C. They are exceptionallysuited for the analysis of traces since very small amounts only of thesubstances are required for a faultless separation.

As further detailed investigations have shown the said layers have avery high Boden-number of theoretical plates (which is known toconstitute a theoretical parameter for the quality of the stationaryphase).

Also in the chromatographic method according to the present inventionthe rate of travelling of the front of the liquid medium obeys thesquare root lay of the height reached by it, as known for paper and thinlayer chromatography. Although it has been found that the total amountof the vapor deposited layer substance has a certain influence on theheight of rising attainable, these factors can easily be kept withinnarrow limits so that layers are obtained which ensure accuratelyreproducible results.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1 diagrammatically shows a sheetconstituting the solid phase dipped in the mobile phase, and rise of thelatter with spots of the substances to be separated, and spots ofcontrol materials.

FIG. 2 is a similar view showing spots of various amounts of substancesto be separated.

FIG. 3 shows diagrammatically an arrangement for the electricalevaluation of the separation of the substances.

FIG. 4 is a graph of resistance value measured by the arrangement ofFIG. 3 plotted against time.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1 Hereinafter someexamples of the invention will be described in more detail. By vapordeposition of indium on a glass plate firstly metal layers were producedwhich subsequently were tempered in a furnace in air at about 400 to 700C. and thus converted into indium oxide. Layers of indium oxide of athickness from 0.6 to 5 u were obtained which in an electron microscopeshowed a rough, wart-like surface structure, the individual excrescenceshaving dimensions in the order of magnitude of 10 cm.

When a glass plate with the indium oxide layer described hereinabove wasdipped into methanol (as the mobile phase), a height of rise of theliquid film covering the glass plate of 1.5 cm resulted after 5 minutes.In spite of this comparatively low rise a faultless separation could beobtained of a mixture of bromine phenol blue and eosin with the use ofmethanol as the liquid medium.

The drawing (FIG. I) shows the result. 1 denotes the level of liquid(methanol) contained in a trough into which the indium oxide layer 2deposited on a glass carrier was dipped. 3 denotes the starting line onwhich small droplets (amount of the sample 10" grams) of a brominephenol eosin mixture was placed, with, for'control, a droplet 5 of pureeosin being placed on the left, and a droplet 6 of pure bromine phenolon the right thereof. After a period of 5 minutes the separation shownin FIG. 1 was reached. The pure eosin travelled up to the mark 5', thepure bromine phenol up to 6', while the mixture of the two substancesproduced the two marks 4' and 4", the lower one of which indicates theeosin component, while the upper one corresponds to the bromine phenolcomponent, as indicated by the conformity with the control mark 5 and6'.

EXAMPLE 2 In a further example according to FIG. 2 four differentamounts (a d) of a l: 1 mixture of bromine phenol blue and eosin wereplaced on a layer of indium oxide of a thickness of 2a, produced in thesame manner as in Example 1. The mobile phase was again methanol. Theperiod of the tests amounted to 2 minutes each. The travel distance ofthe bromine phenol amounted to 1 cm.

It is a surprising fact that by the use of the indium oxide layerdescribed on this short travel path the same separation efiect wasattained as hitherto in the thin-layer chromatography with a travel pathof 10 cm. This shows clearly that with the new method considerablespace-saving can be attained. and that substantially smaller amounts ofsubstances are needed. FIG. 2 shows the chromatograph of the fourdifferent amounts of samples of the aforesaid mixture of dyes. It issurprising that even the amount of substance (d) of only 2.109 gramsproduces an unequivocal and clear separation.

Apart from layers of indium oxide also layers of bismuth oxide and tinoxide are well suited for carrying out the invention, which layers canbe produced in the same way as described for the indium oxide layers byvapor deposition of the metal and subsequent oxidation. Similar althoughless pronounced effects can be attained with any other layers of roughsurfaces. (Conf. Example 5).

The method according to the invention is naturally not confined to theseparation of dyes. The examples of dyes have the advantage that theseparation effect can be observed without special aid. Obviously all thedetection methods used in other thin-layer chromatography methods may beused, e.g. those based on ultra-violet absorption, fluorescence,variation of the refractive index or dyeing by chemical means.

A particularly favorable method of detection for chromatographs producedaccording to the invention will be explained in more detail withreference to FIG. 3.

EXAMPLE 3 By a method analogous to that of Example 1 a tin oxide layerof a thickness of some 11. was produced by the vapor deposition of tinand subsequent annealing in air. When treating a mixture of ethylacetate/methanollNl-l of the ratio 50 l 4 as the mobile phase, whichproduced a height of rise of 6 mm in 3 minutes, one attained with such alayer a very good separation of eosine from butter yellow (p-dimethylamino azobenzene).

For characterizing the effectiveness of the method according to theinvention hereinafter the Rf-values will be stated. The Rf-value definesthe ratio of travel path of one of the substances to be separated to thetravel path of the mobile phase used. In the Example 3 an RF value of0.75 was attained for eosine, and of 0.45 for butter yellow.

EXAMPLE 4 Separation of Cs from Co Separation was carried out on a layerof 1:1 0, (as in Example l). The cations to be separated existed assulphates in aqueous solutions, namely as Co and Cs in concentrations ofabout 10 ppm and 20 ppm, respectively. The aqueous solution was mixedwith some wetting agent for improving the spreading when applying thesample. The sample was applied at a distance of 4 mm from the lower edgeof the plate by the aid of a fine capillary. The amount applied amountedto about I 10 grams of Co and 2 10' grams of Cs. The plate was placedinto a small rack which assured a constant depth of dip, and was placedinto the mobile phase.

The depth of dipping amounted to 2 mm, the mobile phase consisted ofethanol (Cl-l OH) and ethyl acetate (CH COOH) at a ratio of 100 l. Theheight of rise amounted to 1 cm, the period to 2 minutes. Measuring ofradio-active spots was made with a scanner at a rate of advance of 2 mmper minute and a time constant of 3 seconds. The recorder and advancefeed ran synchronously. A Geiger-Muller counter tube served as thecounting instrument; good separation resulted with the followingRf-values Rf Co 0.35 RfISTCIl': EXAMPLE 5 An aluminum foil was used as abase the surface of which was covered by a layer of aluminum oxide,which could be reinforced in a known manner by anodizing or etching,whereby surfaces of good spreading characteristic are obtained. To suchan aluminum oxide surface again a test mixture of eosine plus brominephenol blue plus butter yellow was applied. With the base describedserving as the stationary phase good separation could be attained withvarious solvents such as methanol.

EXAMPLE 6 In this case the method according to the invention was carriedout on a surface produced in the following manner:

a. An aluminum foil (of a thickness of 0.2 mm) was etched for 15 minutesin 5% NaOl-l whereby hydrogen was liberated and the formerly uniformlyblanc surface was rendered slightly matt. Cooling was efiected withflowing water.

b. The foil thus pre-etched was anodized with direct current in a bathof 10 percent oxalic acid and 0.1 percent of chromic acid. The anodesheet metal is located in the middle between the two cathodes at adistance of 5 cm on each side. The voltage was 30 40 V direct current;the current density amounted to about 1 arnp/( 10cm); the period ofelectrolysis was 1 to 2 hours, rinsing was effected with flowing water.

c. The oxidized layer was copper-plated in a solution of CuCl; in 5percent hydrochloric acid. This layer was suspended as a cathode betweentwo Cu anodes with a spacing of 5 cm on each side. The copper wasdeposited in the form of grains adhering to the layer. The voltageamounted to 5 to 20 volts direct current; current density 0.2 to 0.5amps/(l0cm)", corresponding to about 0.1 to 0.2 grams of Cu per 10cm).Finally the foil was rinsed with water and acetone and dried at roomtemperature.

On a surface produced like that, e.g., separation of a mixture ofeosine, bromine phenol blue and butter yellow can be effected. For thispurpose the mixture is applied with the aid of thin glass capillaries tothe surface of the foil a 0.1 percent solution, amount applied about0.1/u] corresponding to about 10 grams). Separation took place withpercent methanol and 5 percent benzene as the solvent.

2 cm in 2 minutes Eosine 1 mm Bromine phenol blue 4 mm (at the center ofeach spot) Butter yellow l8 mm Height of rise: Separation effect:

The spots were proved in spectrometer by extinction.

E X A M P L E 7 As in the Example 6, first an aluminum foil was oxidizedand electro-plated with a copper layer. Subsequently a layer of indiumwas applied. The following electrolyte was used:

In:( SO.) 40 grams Na,SO, 3 grams NaCl 1.5 grams H 0 300 grams Afterbeing rinsed with distilled water and ethanol this layer was dried at Cin a drying cabinet.

For the base with indium coating thus produced the following resultswere found:

With ethyl acetate as the solvent:

Height of rise: 30 mm after 30 seconds Rf values: Bromine phenol blue0.05 Eosine 0.05 Butter yellow 0.95

With ethy lacetate plus 2% acetic acid Height of rise: 30 mm after 30seconds Rf values: Bromine phenol blue 0.05 Eosine 0.75 Butter yellow0.95

(l) Eosln-NHc Br IIir O HO 0 Br Br --co0 mn (2) Bromine phenol blue Br-OQVIM B r B r (3) Bromine thymol blue (4) Phenol red llonzyl orange (6)Dlmethyl yellow (butter yellow) C II;

(10) Phenylalanin:

Louein:

10 (13) Red No. 2 (True red E) (14) Red No. 6 (Scarlet GNl E 1'35 1h 0 Hl H3 -N=N S N 03 a k S OaNa (16) Red No. 1 (A20 rubin S) E 122 OH I 5OaNa (16) Black No. i (Brilliant black BN) E 51 OH NH-CO-CH:

S 03N8 (17) Red No. 4 (Cochenille red A) E 124 (18) Blue No. 2(Indigotin I, 15) E 132 K 0 00 IL moss U-somm o=c v 1'1 (19) OreeinumC29HzlNzO7 (non-homigeneous lichen dye, main constituent=0rcln) (20)Acidum caminicum (Carminie acid) Cochenllle dye H, 0 0H 1 ll OQHIIOI HOOH The separations were carried out (unless otherwise stated) on ln Olayers. For this purpose plates of the size 45 45 mm were provided ontheir sides with millimeter-scales in pencil. The origin (null point)was placed about 4 mm above the lower edge. The dyes were mostly inapproximately 0.1 percent solutions in methanol or methanol-water 75 25.The application of the samples was made by means of a fine glasscapillary at distances of about 3 mm to the right and left from the nullpoint. The spots applied had diameters of 0.5 to 1 mm which correspondedto about 2 to 8 10- grams of the substance.

50 milliliters of a suitable solvent were placed into a commerciallyavailable stand cylinder for this-layer chromatography, and left therestanding for some hours for saturation. Then the plate with the samplesapplied was dipped in by means of a dipping device so far that thesolvent level was about 2 mm below the null point. The rise of themobile phase could be observed best obliquely from above, with themobile phase serving as the background. The average periods of riseamounted to about 2 minutes, the heights of rise to 3 to 10 mm. Afterseparation, the plate was taken out, and its lower margin freed fromadhering drops of the solution.

For regeneration, the plates were firstly rinsed with hot water, thenwith alcohol and acetone, and finally tempered for 3 hours at 500 C. Theresults are compiled in the Tables 1, 2 and 3.

TABLE 1 Separation of Indicator Dyes Example Mobile Phase Test Rf Number(Height of rise) 8 Methanol l 0.50

(2) 0.69 9 Methanol p.a. l) 0 :Acetonitril (2) 0.6 1:1 (3) 0.7 (4 mm) 10Methanol p.a. (l) 0 (3 mm) (2) 0.65 (3 0.75 (4) 0.5 Methanol: l 0Acetonitril: l 0.3 2 Methylenchloride: (2) 0.62 Ammonia (3) 0.7520:20:20:0.2 (4) 0 3 mm) (4) 0.45 12 Methanol p.a. (2) 30 mm l3 Ethylacetate l) stationary 0.5 Methanol: 1) Phase 0.7 Ammonia (6) Bi 0 0.950:10:02 mm) TABLE 2 Separation of Dyes for Food Stuff Example MobilePhase Test R, Number (Height of rise) 14 Acetone-Methanol l3) 0. 3 WaterM) 0.8 80:20:10

l 5 Acetone-Methanol- 15) 0.5

Water l 6) 0.05 l 0: l0 (6 mm) 16 Acetone- (17) 0.65 Benzene-Water 18)0.15 8O: l0: l0 l7 Methanol p.a. 19) 0.6 6 mm) (20) 0.0 18Methanol-water 19) 0.7

(20) 0.05 65: l 5 6 mm) l 8 0.5 19 Methanol- (19) 0.92 Ethyl acetate 18)0.41 Water 16) 0.25 70:30:15 20) 0.05 (6 mm) TABLE 3 Separatio n ofOestrogens and Amino Acids Example Mobile Phase Test R, Number (Heightof rise) 20 Acetone (7) 0 (3 mm) (3 0.6 2 l Acetone: (7) 0.55

Methanol (8) 0.7 l 0: 1 (5 mm) 22 Acetone: 7) 0.33

Methanol (8 0.6 20: l (5 mm) 23 Ethyl acetate (7) 0 3 mm) 8 0.75 24Methanol (9) 0 (5 mm) l 0) 0.3 25 Methanol: 1 l) 0 Buthanol: 12) 0.3Acetum glaciale 10: 10.3 5 mm For the oestrogens: a l;l mixture ofpotassium hexacyanoferrate (ill) with a 2% solution of iron ("1)-chloride a 0.3% solution of ninhydrin in ethyl alcohol p.11.Subsequently were developed at l 10C.

For the amino acids:

In HO. 3 a new arrangement is illustrated by means of which theseparation of substances can be proved in a simple way. Therein 31denotes a glass plate dipping into a solvent 32 for the substances to beascertained, as shown. 33 denotes a strip of an indium oxide layerapplied to the said glass plate, which upon the rise of the liquidprescribes the path thereof. 34 and 35 are two electrodes laterallycontacting the strip of indium oxide, which may be produced by vacummdeposition of metal in vacuo. The two electrodes are connected via acurrent measuring instrument 36 to a source 37 of constant voltage, sothat the value of the current constitutes a measure of the resistancebetween the electrodes 34 and 35.

When in performing the method according to the invention .a sample isplaced on the point 38 of the layer the substance curve 41 is obtained,as shown for example in FIG. 4; the abscissae 42 denote the time t, andthe ordinates 43 the re-- sistance R.

Instead of that arrangement of the electrodes 34, 35, in which theelectric resistance in the direction transversely of the strip 33 of thechromatographic layer is measured, the electrodes could be arranged onebehind the other along the strip, which electrodes would measure theresistance in the longitudal direction. For this purpose metal strips 45and 46 could be used for example, which consist of strips of metal of awidth of a tenth of a millimeter vapor deposited on the layer of indiumoxide, as indicated in FIG. 3, without disturbing the .chromatograph.The spacing of these strips contributes to deterrning the dissolvingpower.

The arrangement illustrated shows the principle only. Obviously morecomplicated resistance measuring circuits may be used as required formeasuring the sometimes very high elec trical resistance of the layersadsorbed.

It is of advantage for the method of detection described last to use asthe mobile phase an electrically non-conductive liquid, and as thestationary phase a solid having an insulating surface. The convenientshape of the electrodes depends on the dissolving power to be attained;the width of the electrode should be as small as possible as comparedwith the width of the bands occurring in a certain case measured in thedirection of movement of the bands.

What we claim is:

l. A method for the chromatographic separation of substances, whereinthe improvement comprises the steps of preparing a non-porous surface ofmicro roughness, and contacting said prepared surface with a liquid thatspreads as a continuous thin film over the surface only, thusconstituting the mobile phase of a chromatographic process.

2. A method as claimed in claim 1, comprising the preparation for use ofa non-porous solid having a rough surface as a stationary phase.

3. A method as claimed in claim 1, comprising the step of freshlypreparing said surface before wetting the surface with said liquid.

4. A method as claimed in claim 1, comprising the step of producing alayer by vapor deposition in vacuo on a base to form said surface as astationary phase.

5. A method as claimed in claim 4, wherein said layer has a maximumthickness of 10"..

6. A method as claimed in claim 4, wherein said layer has a surfaceroughness of the order of magnitude of 0.l to 114..

7. A method as claimed in claim 4, comprising the steps of producingsaid layer by vapor deposition of a metal. and subsequently oxidizingthe same.

8. A method as claimed in claim 4, comprising the steps of vapordeposition of indium and subsequent oxidation thereof to fonn a layer ofindium oxide.

9. A method as claimed in claim 4, comprising the steps of vapordeposition of bismuth and subsequent oxidation thereof to form a layerof bismuth oxide.

10. A method as claimed in claim 4, comprising the steps of vapordeposition of tin and subsequent oxidation thereof to form a layer oftin oxide.

11. A method for the chromatographic separation of substances whereinthe improvement comprises the steps of preparing a non-porous surface ofmicro roughness, and contacting said prepared surface with a liquid thatspreads as a continuous thin film over the surface only and pastelectrodes, and continuously measuring the electric resistance betweensaid electrodes, said liquid constituting the mobile phase of achromatographic process.

12. A method as claimed in claim 11, wherein an electricallynon-conductive liquid is used as the mobile phase, and said surface isan electrically insulating surface used as a stationary phase.

13. A method as claimed in claim 11, comprising the step of forming saidelectrodes by vapor deposition of a metal on said solid.

2. A method as claimed in claim 1, comprising the preparation for use ofa non-porous solid having a rough surface as a stationary phase.
 3. Amethod as claimed in claim 1, comprising the step of freshly preparingsaid surface before wetting the surface with said liquid.
 4. A method asclaimed in claim 1, comprising the step of producing a layer by vapordeposition in vacuo on a base to form said surface as a stationaryphase.
 5. A method as claimed in claim 4, wherein said layer has amaximum thickness of 10 Mu .
 6. A method as claimed in claim 4, whereinsaid layer has a surface roughness of the order of magnitude of 0.1 to 1Mu .
 7. A method as claimed in claim 4, comprising the steps ofproducing said layer by vapor deposition of a metal, and subsequentlyoxidizing the same.
 8. A method as claimed in claim 4, comprising thesteps of vapor deposition of indium and subsequent oxidation thereof toform a layer of indium oxide.
 9. A method as claimed in claim 4,comprising the steps of vapor deposition of bismuth and subsequentoxidation thereof to form a layer of bismuth oxide.
 10. A method asclaimed in claim 4, comprising the steps of vapor deposition of tin andsubsequent oxidation thereof to form a layer of tin oxide.
 11. A methodfor the chromatographic separation of substances wherein the improvementcomprises the steps of preparing a non-porous surface of microroughness, and contacting said prepared surface with a liquid thatspreads as a continuous thin film over the surface only and pastelectrodes, and continuously measuring the electric resistance betweensaid electrodes, said liquid constituting the mobile phase of achromatographic process.
 12. A method as claimed in claim 11, wherein anelectrically non-conductive liquid is used as the mobile phase, and saidsurface is an electrically insulating surface used as a stationaryphase.
 13. A method as claimed in claim 11, comprising the step offorming said electrodes by vapor deposition of a metal on said solid.